Retinal ganglion cells and Mller glia are particularly susceptible to mechanical forces which drive inflammatory activation and RGC degeneration in diseases such as glaucoma, but the pressure transduction mechanisms are not well understood. Earlier studies have been limited to phenotyping the genetic, molecular, cellular and behavioral consequences of RGC injury and glial activation induced by elevated pressure. While many biochemical pathways were shown to be altered in hypertensive eyes, the molecular sensors that transduce mechanical forces remain obscure, confounding interpretations of time-dependence of pressure-induced remodeling changes within the retina. The dominant hypotheses about pressure injury in glaucoma focus on the role of forces on the stretch of the lamina cribrosa yet mice develop the disease but do not have the collagenous lamina. The axocentric hypotheses also cannot explain how mild pressure elevations induce early changes in dendritic architecture and synaptic function, or activate glia without visible changes in axonal transport. It is also not known how physiological levels of intraocular pressure might inform RGC physiology and whether they are sufficient to integrate with the synaptic (light) responses. Finally, although glia are often the earliest responder to mechanical stress, the mechanisms that impel mechanosensitivity to these cells and how they impact RGC physiology remain largely unknown. The proposed work addresses these confounds by identifying the mechanotransducers and elucidating their role in RGC and Mller glial calcium homeostasis and polymodal integration of pressure into the (patho)physiological retinal response. The project tests the central hypothesis that pressure sensitivity of dendrites, somata and axons of RGCs and glia is governed by mechanosensitive ion channels, which maintain tensile homeostasis and modulate calcium homeostasis, excitability and gliotransmitter release in response to changes in ocular pressure or strain. Leveraging the recently derived data and using novel mechanobiological tools, Aim 1 will identify and characterize mechanosensing ion channels in the RGC plasma membrane, quantify their activation by pressure and matrix stretch, and test the hypothesis that mechanical strains are transmitted from the plasma membrane into the cell interior through the cytoskeleton. In Aim 2 we propose to characterize the polymodal mechanism through which mechanical stimuli are integrated with the effects of temperature and synaptic (light) responses, and to test a novel hypothesis regarding the regulation of RGC tensile homeostasis. Aim 3 will characterize the molecular mechanisms whereby mechanically induced glial activation influences RGC physiology, thus providing insight into the early inflammatory mechanisms in diseases such as glaucoma. Taken together, the proposed studies may deepen our understanding of retinal function by uncovering new mechanisms that respond to acute and chronic mechanical forces and by reconciling currently disparate hypotheses about retinal pressure transduction. In addition, these studies will aid in the understanding of neurodegeneration that is required to optimize early diagnosis and neuroprotective treatment, which are currently lacking in glaucoma. During the last few years, mutations in putative mechanosensing ion channels have been shown to cause many human diseases and disorders, including severe dysplasias, gliovascular abnormalities and axonal neuropathies but their impact on visual signaling is unknown due to the absence of basic studies. The information provided by these studies may thus contribute insights into mechanosensitive mechanisms that underlie retinal disease as well as transduction of mechanical stress within the CNS.